ORIGINAL RESEARCH
published: 17 June 2016
Edited by:
Sergey Shabala,
University of Tasmania, Australia
Reviewed by:
Lars Hendrik Wegner,
Karlsruhe Institute of Technology,
Germany
Claudio Lovisolo,
University of Turin, Italy
*Correspondence:
Dany P. Moualeu-Ngangue
moualeu@gem.uni-hannover.de
Specialty section:
This article was submitted to
Plant Biophysics and Modeling,
a section of the journal
Frontiers in Plant Science
Received: 29 February 2016
Accepted: 03 June 2016
Published: 17 June 2016
Citation:
Moualeu-Ngangue DP, Chen T-W and
Stützel H (2016) A Modeling Approach
to Quantify the Effects of Stomatal
Behavior and Mesophyll Conductance
on Leaf Water Use Efficiency.
Front. Plant Sci. 7:875.
doi: 10.3389/fpls.2016.00875
Frontiers in Plant Science | www.frontiersin.org
doi: 10.3389/fpls.2016.00875
A Modeling Approach to Quantify the
Effects of Stomatal Behavior and
Mesophyll Conductance on Leaf
Water Use Efficiency
Dany P. Moualeu-Ngangue 1*, Tsu-Wei Chen 1, 2 and Hartmut Stützel 1
1
Vegetable Systems Modelling Section, Institute of Horticultural Production Systems, Leibniz Universität Hannover,
Hannover, Germany, 2 UMR759 Laboratoire d’Ecophysiologie des Plantes sous Stress Environnementaux, Institut National de
la Recherche Agronomique, Montpellier, France
Water use efficiency (WUE) is considered as a determinant of yield under stress and a
component of crop drought resistance. Stomatal behavior regulates both transpiration
rate and net assimilation and has been suggested to be crucial for improving crop WUE.
In this work, a dynamic model was used to examine the impact of dynamic properties of
stomata on WUE. The model includes sub-models of stomatal conductance dynamics,
solute accumulation in the mesophyll, mesophyll water content, and water flow to the
mesophyll. Using the instantaneous value of stomatal conductance, photosynthesis,
and transpiration rate were simulated using a biochemical model and Penman-Monteith
equation, respectively. The model was parameterized for a cucumber leaf and model
outputs were evaluated using climatic data. Our simulations revealed that WUE was
higher on a cloudy than a sunny day. Fast stomatal reaction to light decreased WUE
during the period of increasing light (e.g., in the morning) by up to 10.2% and increased
WUE during the period of decreasing light (afternoon) by up to 6.25%. Sensitivity of
daily WUE to stomatal parameters and mesophyll conductance to CO2 was tested for
sunny and cloudy days. Increasing mesophyll conductance to CO2 was more likely to
increase WUE for all climatic conditions (up to 5.5% on the sunny day) than modifications
of stomatal reaction speed to light and maximum stomatal conductance.
Keywords: Light regulation, water use efficiency, sap flow dynamics, stomatal behavior, mesophyll conductance
INTRODUCTION
Stomata on the leaf surfaces of higher plants control the fluxes of gases between the atmosphere
and the leaf mesophyll. Regulation of stomata by guard cells determines the amount of CO2
available for photosynthesis (A) and the amount of water loss through transpiration (E; Lawson
et al., 2014). To model the relationship between net assimilation rate and stomatal conductance,
several approaches have been developed in the literature. A strong correlation between stomatal
conductance and net photosynthesis (Anet ) is often observed (Wong et al., 1979; Ball et al., 1987)
and modeling this relationship has been attempted (e.g., Hari et al., 1986; Lloyd, 1991; Arneth
et al., 2002; Katul et al., 2009; Medlyn et al., 2011; among others; see also Damour et al. (2010) for
review of stomatal models). Most of the proposed models of stomata only capture the steady state
stomatal conductance, which may occur only exceptionally in natural environments. However, a
1 June 2016 | Volume 7 | Article 875
Moualeu-Ngangue et al.
steady state model is not suitable for evaluating continuous
changes in Anet and E which define water use efficiency (WUE;
Anet /E, WUE, Hubick et al., 1986).
WUE is one of the traits considered as determinant of
yield under water limited conditions and even as a component
of crop drought resistance (Blum, 2009). Several studies have
reported considerable variations in WUE among crop species
(see Ehdaie et al., 1991 and references therein). For steady
state conditions, WUE can be determined by gas exchange
measurements. However, fluctuations in climatic conditions lead
to changes in stomatal conductance and consequently different
instantaneous variations of E and Anet . Therefore, WUE varies
with fluctuating climate conditions during the course of the
day and stomatal behavior. Stomatal behavior is characterized
by the speed of aperture/closure and the initial time lag for
stomatal response to light stimuli, and related to stomatal
morphology, e.g., stomatal size and density (Lawson and Blatt,
2014). For example, modifying stomatal density and stomatal
aperture speed might increase chloroplastic CO2 concentration
(Cc ) and therefore WUE (Merlot et al., 2002; Schlüter et al.,
2003; Büssis et al., 2006; Lawson and Blatt, 2014). However, the
effect of increasing stomatal density on WUE is still difficult
to evaluate and it is still unclear if stomatal speed or stomatal
density might be more beneficial for WUE because the magnitude
of these effects cannot be easily quantified by experiments.
Technically, it is difficult to monitor WUE dynamics due to the
variation of stomatal conductance to water vapor (gsw ) related
to environmental factors. Mathematical modeling of stomatal
dynamics was proposed to assess the effects of varying conditions
on stomatal behavior (Vialet-Chabrand et al., 2013). The model
of Vialet-Chabrand and colleagues includes a steady state target
function and parameters quantifying stomatal speed and initial
time lag for stomatal response. Replacing the steady state target
function by a model of stomatal conductance (e.g., Medlyn et al.,
2011) may allow us to quantify the influence of stomatal behavior
on Anet and E under fluctuating climatic conditions.
Mesophyll CO2 conductance (gm ) is a limiting factor for
CO2 diffusion into the chloroplast and represents therefore a
limitation for net assimilation with a comparable magnitude
with stomatal conductance (Flexas et al., 2008). Although the
relationship between gm and WUE under drought stress is still
subject to discussion (Hommel et al., 2014), changes in gm were
interpreted as adaptation strategy for plants under stress (Warren
and Adams, 2006; Aranda et al., 2007). Therefore, genotypes
with higher WUE were found to have higher gm . In contrast
to stomatal conductance which influences both water loss and
carbon gain, the effect of mesophyll conductance might be
predomiantly on Anet , thereby, increasing WUE (Flexas et al.,
2008).
The aim of this study was to investigate the influence of
stomatal behavior and mesophyll CO2 conductance on daily
WUE for leaves of a cucumber plant grown under non-stress
conditions using a modeling approach. The model includes the
Farquhar-von Caemmerer-Berry model of C3 photosynthesis
(Farquhar et al., 1980), a steady state target stomata model
(Medlyn et al., 2011), dynamics of stomatal reactions to
environmental stimuli (Vialet-Chabrand et al., 2013), water
Frontiers in Plant Science | www.frontiersin.org
Relating Stomatal Behavior to WUE
transport in the leaf (Guyot et al., 2012; Sack and Scoffoni,
2012; Scoffoni et al., 2012; Caldeira et al., 2014; Tardieu et al.,
2015), and a transpiration model (Steppe et al., 2006; Jones,
2013). This model was used to test the following hypotheses: (1)
faster stomatal regulation results in a higher WUE, (2) higher
stomatal density (quantified by increasing maximum stomatal
conductance) leads to a decrease of WUE for all light conditions,
and (3) higher mesophyll CO2 conductance increases WUE.
MATERIALS AND METHODS
Model Description
Variation in mesophyll water content [Wm (t), mol H2 O m−2 ] at
time t is modeled as the difference between water influx [Fi (t),
mol H2 O m−2 s−1 ] and efflux [FO (t), mol H2 O m−2 s−1 ]:
dWm
= Fi (t) − FO (t) . (1)
dt
At the leaf level, FO (t) can be assumed to equal the transpiration
rate [E(t), mol H2 O m−2 s−1 ] (Jones, 2013). Fi (t) depends on
the difference of water potential between the xylem and the
mesophyll (Steppe et al., 2006; Jones, 2013):
Fi (t) = kx (ψx − ψm (t)) , (2)
where ψ x and ψ m (t) are the water potentials (MPa) of xylem and
mesophyll, respectively, and kx (mol H2 O m−2 s−1 MPa−1 ) is the
xylem and mesophyll, determined by water transport pathways
through multiple components, environmental conditions and
time of day (Guyot et al., 2012; Sack and Scoffoni, 2012; Scoffoni
et al., 2012; Caldeira et al., 2014; Tardieu et al., 2015). For
example, the circadian rhythm of hydraulic conductance has a
peak in the early morning (Caldeira et al., 2014; Tardieu et al.,
2015). The hydraulic conductance kx (mol H2 O m−2 s−1 MPa−1 )
is modeled as sum of a radiation dependent and a water potential
and oscillation dependent component (Caldeira et al., 2014;
Tardieu et al., 2015) by:
kx = cvf1 ∗ (kx,0 + kx,C ), (3)
Where
PPFD
 
kx,0 = a + b min ,1 , (4)
PPFDlim
and
5π
 
π
kx,C = τC cos − t + ∗ (ψx − ψm,r ), (5)
60 2
where kx,0 (g H2 O m−2 s−1 MPa−1 ) is the component of xylem
hydraulic conductance that depends on irradiance, a and b
are empirical constants (g H2 O m−2 s−1 MPa−1 ). A constant
water potential in the xylem sap flow is assumed (ψ x =
−0.08 MPa). kx,C (g H2 O m−2 s−1 MPa−1 ) is the oscillation
dependent component of the hydraulic conductance. τC =
0.324/15 g H2 O m−2 s−1 MPa−2 is the sensitivity to the amplitude
of ψx , cvf1 = 0.05556 mol g−1 H2 O is the conversion factor from
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Moualeu-Ngangue et al. Relating Stomatal Behavior to WUE

g to mole H2 O and ψ m,r = −0.9 MPa is the mesophyll reference where hr is the relative humidity of the ambient air (%), a1 =
water potential. PPFDlim = 1000 µmol photon m−2 s−1 is the 0.61375 kPa, a2 = 17.502, and a3 = 240◦ C. The value of −273.16
limit PPFD for the plant leaf. Parameters a and b were estimated is required for the conversion from ◦ K to ◦ Celsius. The slope
using the literature (Caldeira et al., 2014; Tardieu et al., 2015). of the curve relating saturated vapor pressure to temperature is
Assuming that the matric and gravitational components of the therefore defined by:
water potential in the leaf are negligible, ψ m (t) can be calculated
by the contributions of solute and hydrostatic pressure to water a2 a3 δe
s= . (13)
potential, ψ s and ψ p , respectively: (a3 + Ta − 273.16)2

ψm (t) = ψs (t) + ψp (t)
Nm (t)
ψs (t) = −cvf2 RTl (t) , (7)
Wm
 
−β 1− WWm_max
m (t)
ψp (t) = αe
where Nm (mol solute) is the total amount of dissolved solutes,
cvf2 = 55.56 mol L−1 H2 O is the conversion factor from liter
to mole H2 O, R = 8.3145 10−3 L MPa mol−1 K−1 is the gas
constant and Tl (t) is the leaf temperature at time t (in K). Wm (t)
is the water content in the mesophyll cell at time t (mol H2 O
m−2 ). The relationship between hydrostatic pressure and relative
cell volume Wm (t)/Wm_max is an approximation deduced from
Steudle et al. (1977), where Wm_max is the maximum water
content of the mesophyll, Wm (t) is the water content in the
mesophyll cell at time t, α (MPa) is the full hydrostatic pressure
and ß is a measure of mesophyll elasticity.
Variation of the total amount of dissolved solutes in the
mesophyll is given by:
dNm
= ci Nxy Fi (t),
dt
where ci (unit-less) is a factor for ion exchange, and Nxy denotes
the solute concentration in the xylem sap (mol solute mol−1
H2 O).
The transpiration rate [FO (t), mol m−2 s−1 ] is modeled as a
function of leaf temperature (Maes and Steppe, 2012; Jones, 2013;
Tardieu et al., 2015):
1 ρa cp (δe + s (Tl (t) − Ta ))
FO (t) = , (10)
rtw λγ
where ρa = 1.205 103 is air density (g m−3 ), cp = 1.005 the heat
or thermal capacity of air ( J g−1 K−1 ), s is the slope of the curve
relating temperature to saturated vapor pressure (kPa K−1 ), Ta is
air temperature (K), λ = 4417 J m−3 is the latent heat of water
vaporization, γ = 0.0665 kPa K−1 is the psychrometric constant.
rtw = 1/gtw (t) is the total resistance to water vapor transport (s
m−2 mol−1 H2 O) and [gtw (t)] is the total conductance to water
vapor transport (mol H2 O m−2 s−1 ):
gbw gsw (t)
gtw (t) = ; (11)
gsw (t) + gbw
where gbw is the boundary layer conductance (mol H2 O m−2 s−1 )
and gsw the stomatal conductance to water vapor. δe is the vapor
pressure deficit (kPa) defined by:
(6) According to Maes and Steppe (2012) the relation between (Tl −
Ta ) and raH = 1/gaH , the resistance to diffusive heat transfer to
air, is given by:
raH rtw γ (Rn − Gi ) − raH ρ a cp δe
(8) Tl − Ta = , (14)
ρa cp (γ rtw + sraH )
where Rn (J m−2 s−1 ) is radiation and Gi (J m−2 s−1 ) is soil heat
flux which is here assumed to be zero since an individual leaf is
considered. The stomatal conductance to water vapor is assumed
to be 1.6 times the stomatal conductance to CO2 as usually used
in the literature (Medlyn et al., 2002, 2011) where 1.6 is the ratio
of the diffusivities of CO2 and water in air. The conductance
to diffusive heat transfer to air is related to the boundary layer
conductance through the relation gaH = gbH /1.15 where 1.15 is
the product of the ratio of the diffusivities of heat and water in
the boundary layer (dimensionless). Vialet-Chabrand et al. (2013)
proposed a dynamic model to describe the temporal response of
stomatal conductance to water vapor, denoted by gsw (mol H2 O
m−2 s−1 ) to a change of irradiance over time:
(9)
dgsw
 
1.6G (t) − r0

= αg ln (gsw (t) − r0 ) (15)
dt gsw (t) − r0
where r0 (mol m−2 s−1 ) is a parameter describing the initial
time lag of gsw after exposure to an environmental stimulus,
α g is a time constant (s−1 ) for increasing or decreasing of gsw
and G is the steady-state target value of stomatal conductance
to CO2 under the current environmental conditions described
by Medlyn et al. (2011) and Chen et al. (2014). Incorporating
stomatal response to leaf water potential as presented by Tuzet
et al. (2003), the steady state target stomatal conductance G is
defined by:

g1

A
G = g0 + 1 + √ fψm = g0 + gscb A (16)
δe Ca
where parameters g0 (mol CO2 m−2 s−1 ) and g1 are
species-specific constants of stomatal conductance (for
cucumber, 0.009 mol CO2 m−2 s−1 , and 3.51, respectively,
see Chen et al., 2014), Ca is the ambient CO2 concentration at the
leaf surface and fψ m quantifies the dependency of G to mesophyll
water potential (Tuzet et al., 2003). fψ m is defined by:
1 + exp (sf ψr )
fψm = , (17)
1 + exp(sf (ψr − ψm )

a 2 (Ta − 273.16)
   
hr ψ r = −0.9 MPa is the reference water potential, sf = 4.9 MPa−1
δe = 1 − a1 exp (12)
100 a3 + (Ta − 273.16) is an empirical sensitivity parameter of the stomatal reaction to

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Moualeu-Ngangue et al.
water potential, A ( µmol CO2 m−2 s−1 ) is the steady-state net
photosynthesis rate, i. e., the minimum of the Rubisco-limited
(Ac , µmol CO2 m−2 s−1 ) or RuPB-regeneration-limited (Aj ,
µmol CO2 m−2 s−1 ) photosynthesis rate (Farquhar et al., 1980):
Vcmax · (Cc − Γ∗ )
Ac = , (18)
Cc + K m
J · (Cc − Γ∗ )
Aj = . (19)
4Cc + 8Γ∗
Here, Γ∗ is the CO2 compensation point in the absence of
dark respiration (43.02 µmol CO2 m−2 s−1 ), Rd is the daytime
respiration rate (µmol CO2 m−2 s−1 ), Vcmax (µmol CO2
m−2 s−1 ) is the maximum rate of Rubisco activity at the site of
carboxylation, and Km is the effective Michaelis-Menten constant
for CO2 assimilation that considers the competitive inhibition by
O2 (711 µmol mol−1 ); J (µmol e− m−2 s−1 ) is the rate of electron
transport and Cc (µmol mol−1 ) is the mole fraction of CO2 ,
which is calculated by:
G + gm
Cc (t) = Ca − Ac (20)
Ggm
where gm is mesophyll CO2 conductance (mol CO2 m−2 s−1 )
and Ca is the ambient CO2 concentration (380 µmol mol−1 ).
Therefore, the values of Ac and G are the analytical solutions
which satisfy Equations (18–20, 16) at the same time. Replacing
Cc from Equation (20) in Equation (18) yields,
G(t) + gm
Vcmax · (Ca − Ac G(t) gm − Γ∗ )
Ac (t) = . (21)
G(t) + gm
Ca − Ac G(t) gm + Km
As shown by Ögren and Evans (1993), the photosystem II
electron transport rate that is used for CO2 fixation and
photorespiration, J, is related to the amount of incident
photosynthetically active irradiance (Iinc ; µmol photons
m−2 s−1 ) by:
κ2LL Iinc + Jmax − (κ2LL Iinc + Jmax )2 − 4θ κ2LL Iinc Jmax
p
J=
2θ
, Measurements
(22)
where Jmax (µmol e− m−2 s−1 ) is the maximum electron
transport rate at saturating light levels, θ is a dimensionless
convexity factor for the response of J to Iinc , and κ 2LL (µmol e−
m−2 s−1 ) is the conversion efficiency of Iinc to J at limiting light.
Because Ca , Γ ∗ , and Km are constant, for simplification we set
p1 = Ca − Γ ∗ and p2 = Ca + Km . For the sake of simplicity, we
also set g0m = g0 + gm . Using Equations (18, 20), the steady-state
net photosynthesis rate is the solution of the following equation:
c3 A3c + c2 A2c + c1 Ac + c0 = 0, (23)
where
c3 = gscb ,
c2 = g 0m − gscb Vcmax − gscb gm p2 ,
c1 = gm p1 Vcmax gscb − g 0m Vcmax − g0 gm p2 ,
c0 = g 0 gm p1 Vcmax .
Frontiers in Plant Science | www.frontiersin.org
Relating Stomatal Behavior to WUE
The RuPB-regeneration-limited photosynthesis rate Aj can be
found combining Equations (16, 19, 20), and rearranging the
expressions using previous notations and k1 = Ca −Γ ∗ and
k2 = Ca + 2Γ ∗ , it follows that Aj satisfies the following equation:
b3 A3j + b2 A2j + b1 Aj + b0 = 0, (24)
where
b3 = 4gscb ,
b2 = 4g0m − Jgscb − 4k2 gm gscb ,
b1 = k1 gm Jgscb − 4k2 gm g0 − Jg0m ,
b0 = k1 gm Jg0 .
Equations (23, 24) are solved simultaneously in order to
determine the value of A. The target photosynthesis rate A always
exists since there is always at least one real solution of Equations
(23, 24). The current photosynthesis rate is determined from the
current stomatal conductance.
Plant Materials for Model Evaluation
Cucumber seeds (Cucumis sativus, cv. Aramon, Rijk Zwaan, De
Lier, Netherlands) were sown on 10 June 2014 in rock-wool
cubes (36 × 36 × 40 mm) in the greenhouse of the Institute of
Horticultural Production Systems, Leibniz Universität Hannover,
Germany (52.5◦ N, 9.7◦ E). Seven days after sowing, seedlings
were transplanted into larger rock-wool cubes (10 × 10 × 6.5 cm)
for another 7 days. Plants were cultivated on rock wool slabs
(Grodan, Grodania A/S, Hedehusene, Denmark), which were
placed on metal gutters. The day/night temperature for heating
was set to 22◦ C day/20◦ C night. Ventilations opened at 24◦ C
during daytime. Each liter of nutrient solution contained 0.5
g Ferty Basisdünger 2 (Planta GmbH, Regenstauf, Germany,
0.9 mM NO− − 2+
3 , 1.5 mM NH4 , 2.8 mM K , 3.0 mM Ca , 0.4 mM
+
2+
Mg , 0.4 mM H2 PO4 , as well as adequate amounts of the
micronutrients) and extra 0.9 g Ca(NO3 )2 was added in the
solution (5.5 mM Ca2+ and 11 mM NO− 3 ) after the first fruit set.
Sap flow data were collected between 30 and 31 June for model
evaluation and between 06 and 07 July 2014 for model validation
on a fully expanded leaf of a well-watered cucumber plant. The
leaf was located at the upper canopy, positioned toward south-
east and not shaded by other leaves. A heat field deformation
sensor (HFD) was installed on the petiole of the leaf to monitor
the relative water flow through the petiole (Hanssens et al.,
2013, 2014) on these days. A quantum sensor (Li190, Li-Cor,
Lincoln, USA) of photosynthetically active radiation (PAR) was
installed next to the measured leaf in order to capture the PAR
intercepted by the leaf. Data of HFD and the quantum sensor
were logged every minute. Average air temperature and relative
humidity of 12 min in the greenhouse were recorded by sensors
installed 2 m above ground, about 2 m from the measured leaf.
Leaf temperatures were measured hourly by an infrared camera
(E60, FLIR Systems INC, Boston, USA). Leaf water potential
(C52-chambers, WESCOR INC, South Logan., USA) and water
content were measured every 2 h from 8h00 to 18h00. Since
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Moualeu-Ngangue et al. Relating Stomatal Behavior to WUE

the measurements of water potential and water content were
destructive, they were taken on four leaves comparable (leaf age,
position, and orientation in the canopy) to the monitored leaf.
Input Data
Climatic data recorded on the 6 and 7 of August 2014,
representing a sunny and a cloudy day, respectively (Figure 1),
were used as input data. Moreover, to test the effect of rhythmic
fluctuation of climatic conditions on WUE, so called “ideal”
sunny and cloudy days were simulated. Variation of temperature
and radiation during the ideal days was modeled using sinusoidal
functions based on Kimball and Bellamy (1986) light and
temperature models. The equation defining the light intensity on
ideal days is given by:
 
π (t − t0 )
I (t) = Im + Ia cos −π ,
p during the sunny and cloudy days were equal to the integrals
where Im is the average light intensity during daytime (139 µmol
photon m−2 s−1 on the cloudy day, 237 µmol photon m−2 s−1
on the sunny day), Ia (µmol photon m−2 s−1 ) is the amplitude
of the oscillation in light intensity (133.435 on the cloudy day,
137.02 on the sunny day), p is the light fluctuation period and
t0 is used to set the initial time point to minimum for the
integration. Temperature equations were defined in a similar way
and a constant lag of 30 min (observed from recorded data) was
kept between light variation and subsequent temperature change.
Parameters of the ideal sunny and cloudy days were chosen to
ensure that the integrals of radiation and temperature during
the sunny day and the cloudy day were equal to the integrals
of radiation and temperature on ideal sunny and cloudy days
(Figure 1). Using measured data of 06–07/08/2014, a hyperbolic
decay function was fitted (r2 = 0.53, P < 0.0001) to describe the
relationship between relative humidity and temperature:
1173.613
hr (t) = , (26)
Ta (t) − 6.3458
which is valid for Ta > 6.3458.
Parameters of the sinusoidal light and temperature models
were fitted so that the integrals of radiation and temperature
(25)
of radiation and temperature recorded on 6 and 7/08/2014,
respectively. Moreover, simulations were also performed with
different sinusoidal time periods to estimate the effects of
fluctuation frequencies on WUE.
Parameter Fitting
The stomata model was parameterized using measurements with
the Li6400-XT portable photosynthesis system (Li-Cor, Lincoln,

FIGURE 1 | Climatic data of the cloudy, sunny, ideal cloudy, and ideal sunny days. (A) PPFD recorded on 06-07/08/2014, (B) temperature data of recorded on
06-07/08/2014, (C) PPFD on ideal sunny and cloudy days, (D) ambient temperature on ideal cloudy and sunny days. The integral of total radiation and temperature
during the sunny and cloudy days are, respectively, equal to the integral of radiation and temperature during the ideal sunny and cloudy days for a solar period of 2,
1 h, 30 and 15 min.

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Moualeu-Ngangue et al.
USA). The leaf was left in darkness for about 30 min, until
stomatal conductance was close to zero. Then, PAR was set to
1300 µmol photon m−2 s−1 and values of stomatal conductance
were logged every 5 s until reaching stability. With these data,
the time constant αg and the initial lag r0 were estimated using
Equation (15). Using data of 30–31 July 2014, the limit light
intensity for conductivity PPFDlim = 1000 µmol photon m−2 s−1
and the reference mesophyll water potential ψm,r = −0.9 MPa
were estimated to agree with the observations. The ideal light
and temperature model parameters representing the average
and the amplitude of light and temperature per oscillation
period were fitted using recorded light and temperature data
in Excel. The relationship between hr and Ta was fitted using
the software Sigmaplot (version 11.0, Systat software GmbH,
Erkrath, Germany).
Sensitivity Analysis
To test our hypotheses, sensitivity of WUE to the time constant
for stomata aperture and closure, α g , initial lag for stomatal
reaction, r0 , maximum stomatal conductance (modeled in
g1 ) and mesophyll CO2 conductance were analyzed. Stomatal
behavior parameters were fixed to estimated values for cucumber
leaves (α g =4.0516 10−3 s−1 , r0 = 2.674 10−3 mol H2 O m−2 s−1 ).
Then, r0 , was changed to 0.0106, 0.0012, and 0.0002 mol H2 O
m−2 s−1 and α g was changed decreasingly to 0.00682, 0.00338,
0.00134, and 0.00109 s−1 (values in range of the estimates in
Vialet-Chabrand et al. (2013)). In each of these scenarios, WUE
was computed per day and per 5 s. The daily WUE was computed
as net assimilation integral divided by the transpiration integral
in the period from 6:00 a.m. to 20:00 p.m. The maximum
stomatal conductance, which should be proportional to stomatal
density, increases with g1 in Equation (16). We changed the value
of g1 from −40 to 90% and analyzed the impact on the daily
WUE. Mesophyll CO2 conductance (gm = 0.3 mol CO2 m−2 s−1 )
was increased from −40 to a 500% to evaluate the effects of gm on
WUE.
“Ideal” light and temperature models (Kimball and Bellamy,
1986) were chosen to represent slow and fast light changing
FIGURE 2 | Diurnal time courses of mesophyll water potential (A) and water inflow to the mesophyll (B) and comparison between simulated and
measured data during a cloudy (first day) and a sunny day (second day). Dots represent measured data of water potential, and dashed lines represent the sap
flow raw data and solid lines are simulations from the model. Climatic data are the two days measurement data presented in Figure 1, and all parameter values are
defined in Tables 1, 2.
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Relating Stomatal Behavior to WUE
scenarios. Ideal day light intensity was defined with sinusoidal
fluctuating functions (day length = 15 h, frequency = 2 h−1 , 1
h−1 , 30 and 15 min−1 ).
RESULTS
The model describes well the decreasing trend of relative
water content during daytime for both the sunny day and
the cloudy day (Figure 2). During daytime, the relative water
content (RWC) dropped down to 83% on the sunny day and
remained at around 92% during the dark period. The diurnal
trend of leaf water potential shows a decreasing trend of water
potential during the day. The diurnal course of RWC was well
reproduced during the first day (data not shown). On the second
day, however, a discrepancy was found between observations
and estimation in the morning. Overall, a linear relationship
(r2 = 0.54, P = 0.0156) was found between observations
and simulations. Observed leaf temperature was higher than the
simulated values and no linear relationship between observations
and simulations was found (r2 = 0.01, P = 0.641; data
not shown).
Simulations agreed with the observed trend during both the
sunny and the cloudy day. Observed raw sap flow data and
simulated water inflow in the mesophyll were correlated with
r2 = 56%. All fluctuations observed in the sap flow dynamics
data were reproduced by the model and the delay observed with
respect to irradiance was mimicked, although a discrepancy was
observed between simulated and observed sap flow dynamics
during the sunny day. As expected, the simulated net assimilation
rate, the transpiration rate and stomatal conductance followed
the course of the sun (Figures 3A–C). Due to the decrease of
ψ m during daytime, solute accumulation in the leaf increased
during daytime and stabilized during the night (Figure 3D). The
model showed a decreasing trend of relative water content during
daytime due to transpiration in the presence of irradiance and
the circadian clock effects on xylem hydraulic conductivity (data
not shown).
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Moualeu-Ngangue et al. Relating Stomatal Behavior to WUE

TABLE 1 | Input and output variables of the model.

Variable Description Type Units Equations Initial value

Fi (t) Water flux into the mesophyll Output mol H2 O m−2 s−1 (2)
kx (t) Hydraulic conductivity between xylem and mesophyll Output mol H2 O m−2 s−1 MPa−1 (3)
kx,0 Irradiance dependent component of xylem hydraulic conductance Input g H2 O m−2 s−1 MPa−1 (4)
kx,C Oscillation dependent component of xylem hydraulic conductance Input g H2 O m−2 s−1 MPa−1 (5)
ψm (t) Water potential in the mesophyll Output MPa (6) −0.08
ψs (t) Osmotic potential in the mesophyll Output MPa (7) −1.28
ψp (t) Hydrostatic pressure in the mesophyll Output MPa (8) 1.2
Wm (t) Water content in the mesophyll cell Output mol H2 O m−2 (1) 16
Nm (t) Amount of solute in the mesophyll Output mol solute m−2 (9) 0.121
F0 (t), Efflux from the mesophyll Output mol H2 O m−2 s−1 (10)
E(t) Evapotranspiration rate Output mol H2 O m−2 s−1 (10)
gtw (t) Total conductance to water vapor transport Output mol H2 O m−2 s−1 (11)
δe(t) Vapor pressure deficit Input kPa (12)
s(t) Slope of the curve relating the temperature to the vapor pressure deficit Input Pa K−1 (13)
Tl (t) Leaf temperature at time t Output K (14) 295.15
Ta (t) Air temperature Input K –
gsw (t) Stomatal conductance to water vapor Output mol H2 O m−2 s−1 (15) 0.02
gsc (t) Stomatal conductance to CO2 Output mol CO2 m−2 s−1 (1.16) 0.0125
Rn (t) Net radiation Input J m−2 s−1 Measured
G(t) Steady-state target under the current environmental condition Output mol CO2 m−2 s−1 (16)
A(t) Steady-state net photosynthesis rate Output µmol CO2 m−2 s−1 (18, 21, 23, 19, 24)
Cc (t) Chloroplastic CO2 concentration Output µmol mol−1 (20)
hr (t) Relative humidity Input – Measured
J(t) Rate of electron transport Output µmol e− m−2 s−1 (22)
Iinc (t) Amount of incident photosynthetically active irradiance Input µmol photon m−2 s−1 Measured

FIGURE 3 | Simulated diurnal time courses of net assimilation Anet (A), stomatal conductance to water vapor gsw (B), transpiration FO (C), and
amount of solute in the mesophyll (D) during the sunny (first day) and cloudy day (second day). Climatic data used are from the 2 days of measurement data
presented in Figure 1, and all parameter values are defined in Tables 1, 2.

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Moualeu-Ngangue et al. Relating Stomatal Behavior to WUE

TABLE 2 | Model parameters description and estimated values.

Parameters Description Unit Value

Nxy Solute concentration in the xylem sap mol mol−1 H2 O 0.0003

ce Na+ exclusion coefficient – 0.2
a Empirical constants relating hydraulic conductivity to the mesophyll water potential g H2 O m−2 s−1 MPa−1 0.0259
b Empirical constants relating hydraulic conductivity to the mesophyll water potential g H2 O m−2 s−1 Mpa−1 0.2268
cvf1 Conversion factor from g to mole H2 O mol g−1 H2 O 0.05556
cvf2 Conversion factor from L to mole H2 O mol L−1 H2 O 55.56
ψ m,r Mesophyll reference water potential MPa −0.9
PPFDlim Limit PPFD for the plant leaf µmol photon m−2 s−1 1000
τC Sensitivity to the amplitude g H2 O m−2 s−1 MPa−2 0.324/15
Wm_max Maximum water content of the mesophyll mol H2 O m−2 16
ψx Water potential in the xylem Mpa −0.08
R Ideal gas constant L Mpa mol−1 K−1 8.314.10−3
α Full turgor pressure Mpa 2.4
β Measure of the mesophyll elasticity – 10.6
ρa Air density g mol−1 28.9645
ci Ion exclusion factor – 2
cp Thermal capacity of the air J g−1 K−1 1.012
γ Psychrometric constant kPa K−1 0.0665
gaW Boundary layer Conductance to water transport mol m−2 s−1 2.7
gaH Conductance to sensible heat transport mol m−2 s−1 gbw /1.15
a1 Empirical constants relating vapor pressure deficit to relative humidity kPa 0.61375
a2 Empirical constants relating vapor pressure deficit to the air temperature – 17.502
a3 Empirical constants relating vapor pressure deficit to the air temperature K 240.97
λ Latent heat of water vaporization J mol−1 H2 O 44172
Gi Soil heat storage J m−2 s−1 0
αg Time constant for the stomatal conductance s−1 0.0040516
r0 Parameter describing the initial time lag mol H2 O m−2 s−1 0.002674
g0 Species-specific constants of stomatal conductance mol m−2 s−1 0.009
g1 Species-specific constants of stomatal conductance 3.51
Γ* Constant CO2 compensation point of assimilation in the absence of dark respiration µmol CO2 m−2 s−1 43.02
Vcmax Maximum rate of Rubisco activity at the site of carboxylation µmol CO2 m−2 s−1 102
Ca Ambient CO2 concentration at the leaf surface µmol CO2 m−2 s−1 380
Rd Dark respiration rate mol CO2 m−2 s−1 1.08
Km Michaelis-Menten constants of Rubisco for CO2 µmol CO2 mol−1 711
gm Mesophyll CO2 conductance mol CO2 m−2 s−1 0.3
Jmax Maximal rate of electron transport µmol e− m−2 s−1 140
θ Convexity factor for the response of J to Iinc – 0.75
κ2LL Conversion efficiency of Iinc into J at low light µmol e− m−2 s−1 0.425

Effects of Stomatal Speed on WUE
Daily WUE was 3.605 and 4.685 mmol CO2 mol−1 H2 O and
on the sunny and cloudy day, respectively. Initial time lag for
stomatal response did not lead to an appreciable change of daily
WUE (<0.5%, figure not shown). Increasing the time constant
for stomatal response slightly increased WUE in all scenarios
(Figure 4). During the cloudy day, an increase of 1.2% in WUE
was reached for an increasing stomatal speed by 60% and a light
period of 2 h (Figure 4A). The daily WUE increased by 1.46% for
the ideal sunny day with a 2 h period of light and temperature
fluctuation (Figure 4B).
When ambient light drops quickly, stomata with slower
opening/closure time need more time to close, and, due to
reduced radiation (Figures 5A, 6A, shaded area), the evaporation
rate (Figures 5D, 6D, shaded area) is higher, and the stomatal
limitation to net assimilation is lower (Figures 5B,C, 6B,C,
shaded area). Therefore, WUE is lower for the slower reacting
stomata (Figures 5E, 6E, shaded areas). The impact on WUE
depends on climatic conditions on that day. In case of constant
light intensity over the day, the stomatal speed did not affect
WUE. A higher stomatal speed instantaneously increased WUE
by up to 6.25% on cloudy day, depending on light variation
(Figures 5E, 6E, shaded areas). The model was tested for different
light exposure scenarios, including extreme cases when PPFD
on sunny days was multiplied by 2, the temperature multiplied
by 1.5 and the relative humidity multiplied by 0.6, and for

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Moualeu-Ngangue et al. Relating Stomatal Behavior to WUE

FIGURE 4 | Effect of varying the time constant for stomata response (α g ) on daily water use efficiency (WUE) during the sunny day (B), the cloudy day
(A), and different ideal sunny (B) and cloudy days (A) scenarios. Simulations were performed per second over 15 h (from 06:00 a.m. to 9:00 p.m.). The speed of
stomatal aperture was varied in a set of 15 values and all climatic data were extracted from the 2 days measurement data presented in Figure 1 and ideal sunny and
cloudy days scenario. The ideal sunny and cloudy days 1 to 3 corresponds to sinusoidal fluctuation frequency equal to 2 h−1 , 1 h−1 and 30 min−1 . All other
parameter values are defined in Tables 1, 2.

different values of boundary layer conductance. Similar results DISCUSSION

were observed for all scenarios (Figures 5E, 6E).
Effects of Increasing Maximum Stomata
Conductance on WUE
An increase of 20% of maximum stomatal conductance lead to
a decrease of WUE of up to 8.66% during the sunny day and
8.57% during the cloudy day (Figure 7). In fact, increasing the
maximum stomatal conductance increased the actual stomatal
conductance and therefore transpiration rate. A higher net
assimilation rate was also obtained in all cases, but the increased
net assimilation did not compensate the water lost, and therefore,
the WUE decreased for all stomatal aperture and closure speeds.
Speed of stomatal response to environment, stomatal density
and mesophyll conductance to CO2 have been proposed to be
important traits of plant to better adapt to drought stress (Merlot
et al., 2002; De Lucia et al., 2003; Warren et al., 2003; Büssis et al.,
2006; Lawson and Blatt, 2014; Franks et al., 2015). Previous work
reported that the speed of stomatal response might be more likely
to enhance WUE than stomatal density (Lawson and Blatt, 2014),
but their effects were not quantitatively assessed. To overcome
the technical difficulties in quantifying the influence of these
traits on daily WUE, a model describing the dynamics of WUE
controlled by stomatal behavior and mesophyll CO2 conductance
under different climatic conditions was presented.

Effects of Higher Mesophyll CO2 Stomatal Speed

Conductance on WUE
Increasing mesophyll CO2 conductance increased daily WUE by
up to 4.5% on the cloudy day and 5.5% on the sunny day, despite
an increase in transpiration rate (1.5%, Figures 8A,C,D,F).
However, increasing gm beyond 0.8 mol m−2 s−1 , had only
negligible effects on WUE for all stomatal speeds. Combining
an increase in stomatal speed and gm slightly increased
WUE, and increased Anet more than the only effect of gm
(Figures 8B,E).
Effects of Fluctuating Irradiance on WUE
Using ideal sunny day and ideal cloudy day scenarios, the light
period was changed from 2 min 30 s to 2 h. The results show that
WUE is maximal when the fluctuation period is around 60 min
(Figure 9). WUE increased by as much as 70% depending on
fluctuation period and total daily radiation.
Although a faster reaction of stomatal conductance can increase
the instantaneous WUE by up to 20% (Figures 5E, 6E), it did not
improve daily WUE (Figures 4A,B). Fast stomatal opening speed
in reaction to light increases carbon gain and water loss through
transpiration at the same time. Therefore, it does not significantly
increase WUE. The result is not in agreement with Lawson and
Blatt (2014) who suggested that fast stomatal response increases
both daily carbon gain and WUE. This discrepancy might be
due to the fact that Lawson and Blatt (2014) calculated the
intrinsic WUE (defined as Anet /gsw ) and ignored the influence
of light energy on transpiration. Moreover, Lawson and Blatt
(2014) argued that a slow stomatal response creates a stomatal
limitation to photosynthesis and inferred that a fast response
to light should reduce this limitation. However, the range of
stomatal conductance from their measured data is 0.05–0.13
mol m−2 s−1 , a range where gsw limitation would be strongest.
In contrast, our gsw data ranged from 0.15–0.30 mol m−2 s−1 ,

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Moualeu-Ngangue et al. Relating Stomatal Behavior to WUE

FIGURE 5 | Effect of varying time constant for stomata response (α g ) on net assimilation rate (B), stomatal conductance (C), transpiration rate (D),
water use efficiency (E) for different photosynthetic active radiation (PAR; A) during the sunny day. Simulations were performed in steps of 1 s over 2 h (from
10:00 a.m. to 12:00 p.m.) and are extracted from measurements over 2 days data. Solid lines represent the fastest stomata opening/closing (αg = 4.052 · 10−3 ) and
dotted lines represent the simulation result for αg = 1.01 · 10−2 . Highlighted areas correspond to time courses where slower reacting stomata have a better water use
efficiency. Other climatic data were extracted from the 2 days measurement data presented in Figure 1, and all other parameter values are defined in Tables 1, 2.

i.e., gsw limitation was much less than in the dataset of Lawson
and Blatt (2014). The model should be calibrated to plants with
lower maximum gsw , to quantify the combined effect of α g and
g1 on WUE.
Effect of g1
Medlyn et al. (2011) demonstrated mathematically that the
biological interpretation of g1 is WUE. This interpretation has
been further proved by a global dataset showing that g1 reduces
with available water in the soil (Lin et al., 2015). Therefore, it is
not surprising that increase of g1 decreased WUE (Figure 7A).
It is interesting to identify the traits determining g1 . According
to Equation (16), g0 and g1 are the physiological parameters
which could increase gsw . Parameter g0 represents the gsw value
in the dark and is normally close to zero (although genotypes
in Arabidopsis with constantly high gsw in the dark have been
found recently; Costa et al., 2015). Therefore, parameter g1
should be the factor determining the magnitude of gsw , which
are related to stomatal size and density. This idea can be
supported by the recent publication showing that reducing
maximal gsw by stomatal density increases WUE (Franks et al.,
2015). Unfortunately, g1 was not estimated in this publication.
Further, study which could show the relationship between g1 and
stomatal density may help us to approach a more mechanistic
understanding on g1 . A minimum decrease of the daily WUE
was 5.1% for an increase of g1 by 20%. This result does not agree
with the instantaneous WUE measured by Franks et al. (2015)
under steady state conditions. In contrast to Franks et al. (2015),

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Moualeu-Ngangue et al. Relating Stomatal Behavior to WUE

FIGURE 6 | Effects of varying the time constant for stomata response (αg ) on net assimilation rate (B), stomatal conductance (C), transpiration rate
(D), water use efficiency (E) for different photosynthetic active radiation (PAR; A) during the cloudy day. Simulations were performed in steps of 1 s over 2 h
(from 10:00 a.m. to 12:00 p.m.) and are extracted from measurements over 2 days’ data. Solid lines represent the slowest stomata opening/closing (α g = 4.052
10−3 ) and dotted lines represent the simulation result for α g = 1.01 10−2 . Shaded areas correspond to the period where slower reacting stomata have a higher water
use efficiency. Other climatic data were extracted from the 2 days’ measurement data presented in Figure 1 and all other parameter values are defined in Tables 1, 2.

our results suggest that increasing the maximum stomatal stabilize for 45 min, and therefore, did not account for the
conductance will improve the net assimilation (more than stomatal behavior under naturally changing environment. Under
12.5%), but may not increase WUE. In fact, Franks et al. constant climatic conditions, a similar result was found (data
(2015) estimated a steady state WUE, by letting gsw and A not shown).

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Moualeu-Ngangue et al. Relating Stomatal Behavior to WUE

FIGURE 7 | Effect of increasing maximum stomatal conductance on daily water use efficiency. g1 is a parameter characterizing the maximum
stomatal conductance. (A) Daily WUE for different values of g1 for a cucumber leaf during the recorded sunny and cloudy days. (B) Total daily transpiration rate (FO )
and daily net assimilation rate (Anet ) for different values of the maximum stomatal conductance. Simulations were performed per second over 15 h (from 06:00 a.m. to
9:00 p.m.). Values of g1 = 3.51 was increased from −40 to 90% and each point on the figure is 1 day simulation result. Other climatic data were extracted from the 2
days measurement data presented in Figure 1 and all other parameter values are summarized in Tables 1, 2.

FIGURE 8 | Combined effect of increasing mesophyll CO2 conductance (gm ) and stomatal speed (α g ) on daily water use efficiency (WUE). (A,D) daily
WUE for different values of gm and αg for a cucumber leaf during the recorded sunny (A) and cloudy (D) days. (B,E) total daily transpiration rate (FO ) during the
recorded sunny (B) and cloudy (E) days for different values of gm and αg . (C,F) total daily net assimilation rate (Anet ) during the recorded sunny (C) and cloudy (F)
days for different values of gm and αg . Simulations were performed per second over 15 h (from 06:00 a.m. to 9:00 p.m.). Values of gm = 0.3 was increased from −40
to 500% and α g was varied in a set of 15 values and the figure present daily simulation result. Other climatic data were extracted from the 2 days measurement data
presented in Figure 1 and all other parameter values are summarized in Tables 1, 2.

Effect of Light Fluctuations period, the stomatal responses and changes in light intensity
Increasing oscillation frequency of light and temperature could may be synchronized in such a way that the light energy is
increase the WUE by up to 70%. The ideal oscillation period for optimally used by the leaf. In fact, stomatal guard cells react in
a maximal WUE was found around 50 min. At this oscillation response to changes in environmental conditions. If the light

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Moualeu-Ngangue et al.
FIGURE 9 | Effect of increasing light and temperature fluctuation frequency on daily (A) water use efficiency (WUE), (B) total daily transpiration rate
(FO ), and daily net assimilation rate (Anet ). Simulations were performed per second over 15 h (from 06:00 a.m. to 9:00 p.m.). Value of the light sinusoidal period
was increased from 1 min 25 s to 2 h and each point on the figure represents one day simulation summarized. All other parameter values are presented in Tables 1, 2.
intensity reaches very fast a high value and drop quickly, then
the stomatal aperture, because of the speed of response, may not
reach the corresponding maximum target value. Therefore, the
plant may not fully make use of the high light intensity due to a
higher stomata limitation. This observation might explain results
found in the literature. For example, the effects of environmental
fluctuations on stomatal behavior were reported as reason for
the limited effects of stomatal density (Lawson and Blatt, 2014).
However, an increase of 70% in WUE for the same integral of
light flux and temperature was unexpected. Further experiments
may help to find an optimal light and temperature pattern leading
to an optimal WUE for different plant species with different speed
of stomatal response.
Effects of Mesophyll CO2 Conductance
The model suggested that WUE can be improved by up to 5.5%
by increasing mesophyll CO2 conductance (gm ). When gm > 0.8
mol m−2 s−1 , further increase in gm did not significantly improve
WUE. It might be explained by the fact that the ratio Anet /gm
became very low in comparison with Ci for gm > 0.8 so that the
chloroplastic CO2 concentration and Ci are almost similar.
The model showed an influence of mesophyll CO2
conductance on E (Figure 8B). This could have been caused by
the fact that the steady state target of stomatal conductance G
is calculated from the estimated value of net Anet as modeled
by Medlyn et al. (2011), which is a function of mesophyll
CO2 conductance. The model can probably be improved by
considering a different stomatal target model, independent
of Anet , but depending directly on environmental conditions
(radiation, temperature, and vapor pressure deficit or relative
humidity).
The model presented in this manuscript considered a fully
expanded leaf and all photosynthetic parameters were taken
constant during the simulation period. The effect of Rubisco
activation and deactivation (Gross et al., 1991), and the age effect
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Relating Stomatal Behavior to WUE
on photosynthetic parameters might allow a wider application of
the model. Another issue for further development of the model
is the canopy WUE, taking into account the effect of leaf age and
canopy architecture.
CONCLUSION
The model presented in this manuscript allowed to quantify the
effects of stomatal behavior and mesophyll CO2 conductance on
the WUE of a cucumber leaf. Combining stomatal dynamics with
the effects of changing climatic condition on photosynthesis and
transpiration rates allows to find that for the case of cucumber
leaves that was analyzed, an increase of stomatal speed will
not lead to an increase of more than 1.5% of the daily WUE
under normal fluctuating light condition. It was also found
that increasing maximum stomatal conductance decrease WUE.
Increasing mesophyll CO2 conductance can lead to an increase of
daily WUE by up to 5.5%. This suggests that increasing mesophyll
CO2 conductance might be more likely to increase WUE than
increasing stomatal density and speed.
AUTHOR CONTRIBUTIONS
DM and TC Developed the model, DM performed all numerical
simulations, DM, TC, and HS discussed the data and wrote the
paper.
ACKNOWLEDGMENTS
The publication of this article was funded by the Open Access
Fund of the Leibniz Universität Hannover. TC thanks Prof. Kathy
Steppe and Dr. Jochen Hanssens at Ghent University, Belgium,
for their instructions in using HFD sensors and Landgard
Stiftung and TASPO Stiftung for their financial support of his
research stay at Ghent University.
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Moualeu-Ngangue et al.
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2016 Moualeu-Ngangue, Chen and Stützel. This is an open-access
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